Rodent and other mammalian hybridomas are one of the primary sources of monoclonal antibodies. However, the development of rodent derived monoclonal antibodies as therapeutic antibodies is often hampered by the immunogenicity of rodent antibodies in humans. Antibody humanization technology is used to reduce immunogenicity triggered by non-human protein sequence in human while preserving antigen binding affinity and specificity.
Most of therapeutic antibodies are immunoglobulin G class molecules (IgG). One IgG molecule comprises two heavy chains and two light chains forming a heterotetramer “Y” shape molecule. IgG has two antigen-binding regions called Fab (fragment antigen binding) and one constant region called Fc (fragment crystalline). Each Fab region is a heterodimer of VH-CH1/VL-CL, where VH and VL of the Fv region are connected to the constant region of the heavy chain and the light chain, via linkers, respectively. These linkers allow the Fv considerable rotational flexibility. Each VH or VL has 3 hypervariable loops known as CDRs (complementarity determining regions) which sit at the tip of the Fv region. Three CDRs on VH or VL are connected by four framework regions (FRs 1-4). CDR residues are the key determinants of the antigen-binding properties of an antibody. Both heavy chain and light chain CDRs together form the antigen binding site. The heavy chain and light chain FRs constitute a scaffold for the antigen-binding site.
Antibody humanization is achieved by grafting CDRs of a rodent antibody onto a “similar” human framework (acceptor) and selecting minimal number of key framework residues (back-mutations) that are manually selected from a rodent monoclonal antibody and incorporated into human acceptor in order to maintain the original CDR conformation. Such methods are known in the art, and include those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567.
Although conventional antibody humanization is conducted according to these general principles, the choice of acceptor human framework(s) for grafting rodent CDRs as well as a minimal set of backmutations that retain optimal presentation of the CDRs while minimizing immunogenicity risk often varies from one antibody engineer to the other and requires a deep understanding of both immunoglobulin sequence/structure and antibody biology. Thus, antibody humanization is often a time-consuming and expensive process that adds significant expense to the development of a therapeutic antibody. Accordingly, there is an urgent need for improved humanization techniques that are more rapid and routine than conventional approaches.